Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by insect pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH 6:S4–S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Höfte and Whiteley classified B.t. crystal protein genes into four major classes (Höfte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242–255). The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992] Bio/Technology 10:271–275). CryV has been proposed to designate a class of toxin genes that are nematode-specific. Lambert et al. (Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996] Appl. Environ. Microbiol. 62(1):80–86) describe the characterization of a Cry9 toxin active against lepidopterans. Published PCT applications WO 94/05771 and WO 94/24264 also describe B.t. isolates active against lepidopteran pests. Gleave et al. ([1991] JGM 138:55–62), Shevelev et al. ([1993] FEBS Lett. 336:79–82; and Smulevitch et al. ([1991] FEBS Lett. 293:25–26) also describe B.t. toxins. Many other classes of B.t. genes have now been identified.
WO 94/21795, WO 96/10083, related U.S. patents, and Estruch, J. J. et al. (1996) PNAS 93:5389–5394 describe toxins obtained from Bacillus microbes, wherein the toxins were purportedly produced during vegetative cell growth. These toxins were thus termed vegetative insecticidal proteins (VIP). These toxins were reported to be distinct from crystal-forming δ-endotoxins. These applications make specific reference to toxins designated Vip1A(a), Vip1A(b), Vip2A(a), Vip2A(b), Vip3A(a), and Vip3A(b). See also Lee et al., AEM vol. 69, no. 8 (August 2003), pages 4648–4657, for a discussion of Vip3 mechanism of action and truncation. There are no known reports of Vip3 proteins having activity against diamondback moths (Plutella xylostella).
Diamondback moths are known to develop resistance to various chemical pesticides, as well as some B.t. Cry toxins such as Cry1Ab, Cry1Ac, and Cry1C. See, e.g., Syed, A. R. (1992), Insecticide resistance in diamondback moth in Malaysia, pp. 437–442, in N. S. Talekar (ed.) Management of Diamondback Moth and Other Pests: Proceedings of the 2nd International Workshop, AVRDC, Taiwan; Shelton, A. M., et al. (1993), Resistance of diamondback moth to Bacillus thuringiensis subspecies in the field, J. Econ. Entomol. 86:697–705; Tabashnik, B. E., et al. (1990), Field development of resistance to Bacillus thuringiensis in diamondback moth, J. Econ. Entomol. 83:1671–1676; Tabashnik, B. E., et al. (1993), Increasing efficiency of bioassays: evaluating resistance to Bacillus thuringiensis in diamondback moth, J. Econ. Entomol. 86:635–644; Tanada, H. (1992), Occurrence of resistance to Bacillus thuringiensis in diamondback moth, and results of trials for integrated control in a watercress greenhouse, pp. 165–173, in N. S. Talekar (ed.) Management of Diamondback Moth and Other Crucifer Pests: Proceedings of the 2nd International Workshop, AVRDC, Taiwan; Zhao, J. Z., et al. (1993), On-farm insecticide resistance monitoring methods for diamondback moth, Acta Agriculturae Sinica 1(1):(in press); Zhu, G. R., et al. (1991), Insecticide resistance and management of diamondback moth and imported cabbage worm in P. R. China, Resistant Pest Management Newsletter 3(2):25–26; Tabashnik, B. E., (1994), Evolution of resistance to Bacillus thuringiensis, Annual Review of Entomology 39:47–49; Metz, T. D., et al. (1995), Transgenic broccoli expressing a Bacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies, Molecular Breeding 1:309–317; Perez, C. J., et al. (1995), Effect of application technology and Bacillus thuringiensis subspecies on management of B. thuringiensis subsp. kurstaki-resistant diamondback moth (Lepidoptera: Plutellidae), J. Econ. Entomol. 88:1113–1119; Shelton, A. M., Jr., et al. (1993), Resistance of diamondback moth (Lepidoptera: Plutellidae) to Bacillus thuringiensis subspecies in the field, J. Econ. Entomol. 86:697–705; Tang, J. D., et al. (1996), Toxicity of Bacillus thuringiensis spore and crystal protein to resistant diamondback moth (Plutella xylostella), Appl. Environ. Microbiol. 62:564–569; Zhao, J. Z., et al. (2001), Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C, Journal of Economic Entomology 94(6):1547–1552; Cao, J., et al. (1999), Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth resistant to Cry1A or Cry1C, Molecular Breeding, 5(2):131–141.
New classes of toxins and genes are described in WO 98/18932. They are distinct from those disclosed in WO 94/21795, WO 96/10083, WO 98/44137, and Estruch et al.